Figure 5.
Figure 5. Sema3E-PlexinD1 signaling down-regulates Rac1 activity, which is medicated by SH3BP1 via its GAP activity. (A) Constitutively active Rac1 blocks Sema3E-induced collapse. Cells were transfected with a constitutively active form of Rac1 (Rac1Q61L) and treated with control ligand or Sema3E. GFP construct was cotransfected to visualize cell morphology. While the addition of Sema3E to cells overexpressing only GFP led to strong cell collapse (arrows), HUVECs transfected with Rac1Q61L were unable to undergo collapse after treatment with Sema3E. Bar, 100 µm. (B) PlexinD1 and SH3BP1 colocalized with Rac1 at the lamellipodia (arrows) of HUVECs. PlexinD1-GFP and SH3BP1-HA were cotransfected in HUVECs and stained with GFP or HA antibodies combined with a Rac1 antibody. The boxed regions are enlarged on the right. Bar, 10 µm. (C) Sema3E induced a down-regulation of Rac1 activity, which was blocked by PlexinD1 or SH3BP1 siRNA. HUVECs treated with Sema3E for 0, 2.5, 5, 10, or 20 min were lysed, precipitated with the PAK Rac1-binding domain, and blotted with a Rac1 antibody. Rac1-GTP, total Rac1, and Tubulin blots are shown. (D) Quantification of a Rac1 activity assay is shown. *, P < 0.05; **, P < 0.005. Error bars indicate SEM. (E) SH3BP1 GAP activity was required for Sema3E-induced collapse. Deletion of the GAP domain of SH3BP1 as well as the GAP activity–defective mutant failed to rescue SH3BP1 siRNA inhibition of Sema3E-induced collapse. Rescue experiments with SH3BP1ΔRhoGAP, SH3BP1-R232A, and SH3BP1-Res (full-length siRNA resistant) constructs are shown. Cell shape, DTAF labeling (green); vector expression, HA antibody (red). Arrows, cell collapse. Bar, 100 µm. (F) The results from D were quantified and the percentage of cell collapse is shown. ***, P < 0.01; n ≥ 3. Error bars indicate SEM. n.s., not significant.

Sema3E-PlexinD1 signaling down-regulates Rac1 activity, which is medicated by SH3BP1 via its GAP activity. (A) Constitutively active Rac1 blocks Sema3E-induced collapse. Cells were transfected with a constitutively active form of Rac1 (Rac1Q61L) and treated with control ligand or Sema3E. GFP construct was cotransfected to visualize cell morphology. While the addition of Sema3E to cells overexpressing only GFP led to strong cell collapse (arrows), HUVECs transfected with Rac1Q61L were unable to undergo collapse after treatment with Sema3E. Bar, 100 µm. (B) PlexinD1 and SH3BP1 colocalized with Rac1 at the lamellipodia (arrows) of HUVECs. PlexinD1-GFP and SH3BP1-HA were cotransfected in HUVECs and stained with GFP or HA antibodies combined with a Rac1 antibody. The boxed regions are enlarged on the right. Bar, 10 µm. (C) Sema3E induced a down-regulation of Rac1 activity, which was blocked by PlexinD1 or SH3BP1 siRNA. HUVECs treated with Sema3E for 0, 2.5, 5, 10, or 20 min were lysed, precipitated with the PAK Rac1-binding domain, and blotted with a Rac1 antibody. Rac1-GTP, total Rac1, and Tubulin blots are shown. (D) Quantification of a Rac1 activity assay is shown. *, P < 0.05; **, P < 0.005. Error bars indicate SEM. (E) SH3BP1 GAP activity was required for Sema3E-induced collapse. Deletion of the GAP domain of SH3BP1 as well as the GAP activity–defective mutant failed to rescue SH3BP1 siRNA inhibition of Sema3E-induced collapse. Rescue experiments with SH3BP1ΔRhoGAP, SH3BP1-R232A, and SH3BP1-Res (full-length siRNA resistant) constructs are shown. Cell shape, DTAF labeling (green); vector expression, HA antibody (red). Arrows, cell collapse. Bar, 100 µm. (F) The results from D were quantified and the percentage of cell collapse is shown. ***, P < 0.01; n ≥ 3. Error bars indicate SEM. n.s., not significant.

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